Many molecular biology applications, such as capillary electrophoresis, nucleotide sequencing, reverse transcription cloning and gene therapy protocols, which contemplate the transfection, transduction or microinjection of mammalian cells, require the isolation of high quality nucleic acid preparations. Quality is a particularly important factor for capillary electrophoresis for all sequencing methods and for gene therapy protocols. Quantity is an equally important consideration for some applications, for example, large scale genomic mapping and sequencing projects, which require the generation of hundreds of thousands of high quality DNA templates.
Extension product quality is crucial to the success of automated dye-labeled dideoxynucleotide sequencing methods, such as those described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., Sanger, E., et al., Proc. Natl. Acad Sci. 74:5463-5467 (1977), and Mierendorf, R. and Pfeffer, D. Methods Enzymol. 152:5556-562 (1987), and is a particularly critical consideration for capillary electrophoresis protocols. The isolation of high quality nucleic acid preparations from starting mixtures of diverse composition and complexity is a fundamental technique in molecular biology.
The advent of demanding molecular biology applications has increased the need for high-throughput, and preferably readily automatable, purification protocols capable of producing high quality nucleic acid preparations. Although recent technological advancements and the advent of robotics have facilitated the automation of sequencing reactions and gel reading steps, throughput is still limited by the availability of readily automatable methods of nucleic acid purification.
The present invention is a method of separating different species of nucleic acid molecules present in a mixture, which can be a solution or a suspension, on the basis of differences in their molecular size. Separation is accomplished by selectively adsorbing target nucleic acid molecules (e.g., targeted or selected for isolation or purification), present in a mixture to a magnetically responsive solid phase carrier such as paramagnetic microparticles. In addition to target nucleic acid molecules the mixture can comprise other components, which include, but are not limited to, other non-target nucleic acid molecules, proteins, cell components, and reagents or chemicals used in methods in which the nucleic acids are processed or used. That is, the mixture can comprise a wide variety of types of molecules, from which target nucleic acid molecules are separated, by the present method on the basis of differences in molecular size. Thus, the invention discloses a method useful for isolating a particular species of nucleic acid molecule from a mixture. The nucleic acid to be isolated (e.g., separated or purified) (referred to as target nucleic acids or target nucleic acid molecules) are separated from the rest of the mixture based on molecular size and through the physical removal of the solid phase carrier to which the target nucleic acid molecules have been adsorbed according to the method described herein.
The present invention provides a method of selectively isolating (e.g., purifying) a species of nucleic acid molecule, based on its molecular size, from a mixture. This embodiment of the invention involves selectively precipitating and facilitating the adsorption of a target species of nucleic acid molecule to the functional group coated surface of a solid phase carrier. Purification of the target nucleic acids is accomplished by applying an external force which results in the removal (e.g., separation) of the solid phase carrier from the mixture in which the carriers have been suspended. In a preferred embodiment, the solid phase carrier is a paramagnetic microparticle and separation is accomplished by magnetic means.
The present invention is useful to isolate, from a mixture from which at least one species of nucleic acid molecule has been selectively removed, one or more additional (e.g., a second, third, fourth etc.) species of nucleic acid molecules which are of a smaller molecular size than the one or more target nucleic acid species which have already been removed from an initial (or starting) mixture by the method described herein. The additional species of nucleic acid molecule targeted for isolation in this additional embodiment remained soluble in the presence of the PEG and salt concentrations used to isolate the larger nucleic acid molecule and, therefore, will still be present in the mixture from which the first target nucleic acid molecule has been removed.
In an alternative embodiment of the instant invention, two or more species of nucleic acid molecule present in the same mixture, which differ in molecular size from each other by at least a factor of two, are separated from each other. The method described herein is used to isolate a particular species (e.g., a target species) of nucleic acid molecules of virtually any size, present in a wide variety of sources, from other nucleic acid molecules which are also present in the mixture. For example, the method disclosed herein can be used to isolate recombinant nucleic acid species, produced in host cells, including selective RNA precipitations based on molecular size, or replicative form DNA produced by a virus during lytic replication from endogenous host cell nucleic acid species. The method can also be used to isolate a particular species of nucleic acid from a solution resulting from a restriction enzyme digestion or an agarose solution containing nucleic acid. Alternatively, the method disclosed herein provides a size selection purification scheme suitable for use after a DNA shearing process (e.g., hydroshearing or sonication), thereby providing an alternative to the more traditional method of gel electrophoresis and band excision which are conventionally used to isolate a species of nucleic acid molecule targeted for purification. The disclosed method also finds utility as a method of separating multiplex PCR products, or as a sequencing reaction detemplating protocol. For example, using the method disclosed herein solid phase magnetically responsive paramagnetic microparticles can be used to selectively remove sequencing products and DNA templates from sequencing samples.
The present invention is also useful to isolate, from a mixture from which at least one species of nucleic acid molecule has been selectively removed, one or more additional (e.g., a second, third, fourth etc.) species of nucleic acid molecules which are of a smaller molecular size than the one or more target nucleic acid species which have already been removed from an initial (or starting) mixture by the method described herein. The additional species of nucleic acid molecule targeted for isolation in this additional embodiment remained soluble in the presence of the PEG and salt concentrations used to isolate the larger nucleic acid molecule and, therefore, will still be present in the mixture from which the first target nucleic acid molecule has been removed.
One embodiment of the instant invention provides a method of selectively isolating a target species of nucleic acid molecule from a host cell lysate which is a mixture of target nucleic acid molecules, non-target nucleic acid molecules and other cellular components. The presence of these other components (e.g., cellular components, reagents or biomolecules) could have an adverse effect on the downstream molecular biology application for which the target nucleic acids are being prepared. For example, the present method is useful to selectively isolate exogenous nucleic acid from endogenous host cell nucleic acid molecules. More specifically, the present method is useful to selectively isolate recombinant DNA, produced by the replication of exogenous DNA in an appropriate host cell, from endogenous host cell DNA and from other host cell components. Exogenous DNA (e.g., recombinant or plasmid DNA) can be introduced into suitable host cells, or their ancestors, by a vector, such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a phage artificial chromosome (PAC), a P1, a cosmid (plasmids with xcex phage packaging sites) or a bacterial plasmid. The exogenous DNA replicates in the host cell and can be isolated from host cell DNA using the method described herein. Initially, host cell (endogenous) DNA is removed through the use of appropriate concentrations of PEG and salt. Subsequently, exogenous nucleic acid molecules targeted for isolation are removed, again through the use of appropriate concentrations of PEG and salt. In this embodiment, as well as in all others described herein, nucleic acid molecules of varying sizes can be removed from a mixture sequentially. That is, all nucleic acid molecules above a certain size cutoff can be removed through the use of appropriate PEG and salt concentrations, with the result that smaller nucleic acid molecules remain in the mixture. This process can be repeated to remove progressively smaller nucleic acid molecules. According to this embodiment, exogenous nucleic acid molecules are selectively isolated from endogenous host cell nucleic acid molecules and other biomolecules present in a host cell lysate. More specifically, this embodiment comprises combining functional group-coated paramagnetic microparticles and suitable concentrations of a precipitating reagent, for example, a polyalkylene glycol, and a salt to result in the facilitated adsorption of endogenous nucleic acid (e.g., host cell genomic DNA) present in the mixture, to the surfaces of the microparticles suspended therein, and the subsequent removal, such as by magnetic means, of the microparticles from the resulting combination. The removal of the microparticle having host cell genomic DNA (e.g., endogenous DNA) adsorbed to its surfaces from the combination results in the concomitant separation of the target nucleic acid selected for isolation from other relatively smaller-sized nucleic acids (e.g., exogenous nucleic acids), which remain in solution, thereby producing a mixture that is enriched for exogenous nucleic acid. The resulting PEG-facilitated adsorption of the precipitated host cell DNA to the functional group-coated surfaces of the paramagnetic microparticles (which can be considered a pre-clearance of host cell DNA) is rapid; it is generally complete within thirty seconds. Exogenous target nucleic acid molecules present in the mixture enriched for relatively smaller-sized nucleic acids can subsequently be isolated from the mixture by performing a second PEG-induced precipitation, according to the method provided herein.
The present invention further relates to a method of isolating an exogenous DNA template (e.g., a plasmid DNA template) suitable for use in either manual or a high-throughput automated sequencing methods. In general terms this method comprises: treating host cells which contain exogenous DNA (e.g., plasmid DNA) with an alkali and detergent (e.g., sodium dodecyl sulphate (SDS)) combination, thus producing a lysed host cell suspension; suspending paramagnetic microparticles which bind DNA (e.g., have an affinity for DNA) in the lysed host cell suspension in the presence of sufficient concentrations of a DNA precipitating reagent, for example, polyethylene glycol, and a salt to selectively precipitate and adsorb host cell DNA (e.g., genomic DNA), but not exogenous DNA, to the surfaces of the paramagnetic microparticles; removing the microparticles having host cell DNA bound thereto from the suspension, preferably by magnetic means, thereby producing a plasmid DNA-enriched supernatant; combining additional paramagnetic microparticles having an affinity for DNA with the resulting plasmid DNA-enriched supernatant; and adjusting the precipitating reagent and/or salt concentration of this supernatant to suitable levels to result in the selective precipitation and adsorption of exogenous (e.g., plasmid) DNA to the microparticles suspended therein. As a result, exogenous (e.g., plasmid) DNA is bound to the microparticles, thereby producing microparticle-bound exogenous DNA.
The purity of the microparticle-bound exogenous DNA can be improved by washing the particle-bound nucleic acid molecules to remove other host cell biomolecules by contacting the microparticles with a high ionic strength wash buffer which dissolves, for example impurities (e.g., proteins, reagents or chemicals) adsorbed to the paramagnetic microparticles, but does not solubilize the adsorbed DNA. As a result, the exogenous DNA targeted for isolation remains adsorbed to the solid phase carrier surface. The washed, particle-bound exogenous DNA template can subsequently be removed from the solid phase carrier by contacting the washed microparticles with an elution buffer which solubilizes the adsorbed DNA, thereby preparing plasmid DNA suitable for use as a DNA nucleotide sequencing template.
In one embodiment, the invention is a readily automatable method of isolating a plasmid DNA template for nucleotide sequencing. The use of paramagnetic microparticles having functional group-coated surfaces as a bioaffinity adsorbent for nucleic acid molecules affords an alternative, and readily automatable, means of molecular separation useful in the design of a solid phase technique for the selective isolation of nucleic acid molecules targeted for isolation. A particular advantage of the disclosed method is that it obviates the necessity of centrifugation and filtration steps and the use of organic solvents. In addition, the ample surface area provided by suitable solid phase carriers allows the volume of the reactions to be reduced, which not only facilitates automation, but which also eliminates the need for subsequent concentration steps which conventionally require the use of a solvent whose presence in the final preparation can adversely effect the quality of the isolated DNA. Thus, the invention offers purification methods which are simple, cost effective, robust and readily amenable to automation.
The present invention also relates to a kit comprising magnetically responsive microparticles having a functional group-coated surface that reversibly binds nucleic acid molecules, at least one binding buffer, a suitable nucleic acid precipitating reagent and salt at concentrations suitable for reversibly binding nucleic acids onto the surface of the microparticle. The kit may additionally comprise preformulated solutions of a host cell lysis buffer, or reagents for the preparation of such buffer, a wash buffer and an elution buffer. The exact compositions of the buffers may vary with the nature of the starting material and the purpose (e.g., the molecular biology application) for which the nucleic acid preparation is being isolated. The kit may further include a magnetic microtiter plate holder specifically designed to optimize the field strength applied to remove the paramagnetic microparticles from the resulting combinations and solutions.
As described herein, Applicants have shown that different species of nucleic acid present in a solution can be isolated, on the basis of their molecular size, through the use of appropriate concentrations of a nucleic acid precipitating reagent, preferably a polyalkylene glycol, and a salt to result in the selective precipitation and facilitated adsorption of a particular nucleic acid species to the functional group-coated surfaces of a suitable magnetically responsive solid phase carrier which functions as a bioaffinity adsorbent for a species of nucleic acid molecule targeted for isolation.
According to the instant invention, one embodiment of the method comprises selectively adsorbing (e.g., non-covalently binding) target nucleic acid molecules in a reversible manner to paramagnetic microparticles having a functional group coated (e.g., carboxyl-coated) surface by preparing a combination comprising a mixture of nucleic acid molecules, polyethylene glycol, salt, and solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules wherein the polyethylene glycol and salt are present in sufficient concentrations to selectively precipitate the target species of nucleic acid molecules. The combination (first combination) is maintained under conditions appropriate for adsorption of the precipitated nucleic acid molecules to the functional group-coated surfaces of the solid phase carriers, thereby producing solid phase carriers having the target species of DNA bound thereto. Isolation of the target species of nucleic acid molecules is accomplished by removing the nucleic acid-coated carriers from the first combination. The solid phase carriers (e.g., paramagnetic microparticles) can be recovered from the first combination, for example, by applying a magnetic field to draw down the paramagnetic microparticles. More specifically, paramagnetic microparticles are preferably separated from solutions using magnetic means, such as applying a magnet field of at least 1000 Gauss. However, other methods known to those skilled in the art can be used to remove the magnetic microparticles from the supernatant; for example, vacuum filtration or centrifugation can be used. The remaining solution can then be removed; leaving paramagnetic microparticles having a particular nucleic acid species adsorbed to their surface. Once separated from the mixture, the isolated nucleic acid species adsorbed to the solid phase carrier can be recovered by contacting the microparticles with a suitable elution buffer. As a result, a solution comprising the target nucleic acid molecules and paramagnetic microparticles is produced. Using appropriate means, for example, magnetic means, the microparticles are subsequently removed from the solution whereby the target species of nucleic acid molecule is isolated from the mixture and a second mixture is produced.
A suitable elution buffer can be water or any aqueous solution in which the salt concentration and polyalkylene concentration are below the concentrations required for binding of DNA onto magnetic microparticles, as discussed above. For example, useful buffers include, but are not limited to, TRIS-HCl, Tris-acetate, sucrose (20%) and formamide (100%) solutions. Elution of the DNA from the microparticles occurs quickly (e.g., in thirty seconds or less) when a suitable low ionic strength elution buffer is used. Once the bound DNA has been eluted, the magnetic microparticles are separated from the elution buffer.
Optionally, impurities (e.g., host cell components, proteins, metabolites or cellular debris) can be removed by washing the paramagnetic microparticles with target nucleic acid bound thereto (e.g., by contacting the microparticles with a suitable wash buffer solution) before separating the microparticle-bound nucleic acid from the solid phase magnetically responsive microparticle. The composition of the wash buffer is chosen to ensure that impurities either bound directly to the microparticle, or associated with the adsorbed DNA are dissolved. The pH and solute composition and concentration of the wash buffer can be varied according to the types of impurities which are expected to be present. For example, ethanol exemplifies a preferred wash buffer useful to remove excess PEG and salt. The magnetic microparticles with bound DNA can also be washed with more than one wash buffer solution. The paramagnetic microparticles can be washed as often as required (e.g., three to five times) to remove the desired impurities. However, the number of washings is preferably limited to in order to minimize loss of yield of the bound DNA. A suitable wash buffer solution has several characteristics. First, the wash buffer solution must have a sufficiently high salt concentration (a sufficiently high ionic strength) that the nucleic acid bound to the magnetic microparticles does not elute off of the microparticles, but remains bound. A suitable salt concentrations is greater than about 0.1 M and is preferably about 0.5M. Second, the buffer solution is chosen so that impurities that are bound to the DNA or microparticles are dissolved. The pH and solute composition and concentration of the buffer solution can be varied according to the types of impurities which are expected to be present. Suitable wash solutions include the following: 0.5xc3x975 SSC; 100 mM ammonium sulfate, 400 mM Tris pH 9, 25 mM MgCl2 and 1% bovine serum albumin (BSA); and 0.5M NaCl. A preferred wash buffer solution comprises 25 mM Tris acetate (pH 7.8), 100 mM potassium acetate (KOAc), 10 mM magnesium acetate (Mg2OAc), and 1 mM dithiothreital (DTT).
In an alternative embodiment the method provides a method of selectively isolating an additional species of nucleic acid molecules from the second mixture (produced by the removal of nucleic acid-coated microparticles from the first combination) wherein the additional species of nucleic acid molecule is of a smaller molecular size than the first target species isolated from the first combination. More specifically, this method comprises the steps of producing a second combination by adding to the second mixture solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules, polyethylene glycol and salt, wherein the polyethylene glycol and salt are present in sufficient concentrations to precipitate the additional target species of nucleic acid molecules. The second combination is maintained under conditions appropriate for the absorption of the additional target species to the functional group-coated surfaces of the solid phase carries, thereby producing solid phase carriers having the additional target species of nucleic acid molecule bound thereto. Isolation is accomplished by removing the solid phase carriers having the additional species of nucleic acid molecules absorbed thereto from the second combination and eluting the additional target species into a suitable low ionic strength solution.
As used herein the terms xe2x80x9cnucleic acidxe2x80x9d and xe2x80x9cnucleic acid moleculexe2x80x9d are used synonymously with the term polynucleotides and they are meant to encompass DNA (single-stranded, double-stranded, covalently closed, and relaxed circular forms), RNA (single-stranded and double-stranded), RNA/DNA hybrids and polyamide nucleic acids (PNAs).
The term xe2x80x9cspeciesxe2x80x9d as it used herein to refer to nucleic acid molecules means a particular subclass, family or type of nucleic acid molecule defined on the basis of a characteristic size. Thus, the members of a xe2x80x9cspecies of nucleic acid moleculesxe2x80x9d are all of approximately equivalent molecular size within a small range of molecule sizes.
As used herein the term xe2x80x9cisolatedxe2x80x9d is intended to mean that the material in question exists in a physical milieu distinct from that in which it occurs in nature and/or has been completely or partially separated or purified from other nucleic acid molecules.
Appropriate starting material includes, but is not limited to, lysates prepared from cells obtained from either mammalian tissue or body fluids, nucleic acid samples eluted from agarose or polyacrylamide gels, solutions containing multiple species of DNA molecules resulting either from a poymerase chain reaction (PCR) amplification or from a DNA size selection procedure and solutions resulting from a post-sequencing reaction. Suitable starting solutions typically are mixtures of biomolecules (e.g. proteins, polysacchardies, lipids, low molecular weight enzyme inhibitors, oligonucleotides, primers, templates) and other substances such as agarose, polyacrylamide, trace metals and organic solvents, from which the target nucleic acid molecule must be isolated.
For example, a suitable starting material can be host cells containing an exogenous nucleic acid (e.g., recombinant DNA, bacterial DNA or replicative form DNA) which is targeted for isolation from host cell chromosomal DNA and other host cell biomolecules. According to the current method, host cells are lysed using known methods, thereby preparing a mixture suitable for use with the method of the instant invention. An alternative starting material appropriate for use with the current invention is an agarose solution. For example, a mixture of nucleic acid can be separated, according to methods known to one skilled in the art (e.g., gel electrophoresis), such as by agarose gel electrophoresis. A plug of agarose containing nucleic acid on interest can be excised from gel and combined with an appropriate buffer, into which the nucleic acid is released by heating the combination to dissolve the agarose plug. The method of the instant invention can also be used to separate a particular species of DNA present in a post-shearing procedure mixture; or to remove a template and primers from a sequencing reaction or to separate PCR primers from the reaction product of a PCR amplification protocol.
As used herein the terms xe2x80x9cselectivexe2x80x9d and xe2x80x9cselectivelyxe2x80x9d refer to the ability to isolate a particular species of DNA molecule, on the basis of molecular size (e.g., host cell chromosomal DNA or exogenous plasmid DNA), from a combination which includes or is a mixture of species of DNA molecules, such as a host cell lysate and other host cell components. The selective isolation of a particular species is accomplished through the use of an appropriate precipitating reagent (e.g., polyalkylene glycol salt) to result in the precipitation and facilitated adsorption of a,.particular DNA species (e.g., characterized on the basis of size) to the surfaces of paramagnetic microparticles.
Suitable precipitating reagents include ethanol, isopropanol and polyalkylene glycols. Appropriate polyalkylene glycols include polyethylene glycol (PEG) and polypropylene glycol. Generally, PEG is used. Suitable PEG can be obtained from Sigma (Sigma Chemical Co., St. Louis, Mo., Molecular weight 8000, Dnase and Rnase fee, Catalog number 25322-68-3) The molecular weight of the polyethylene glycol (PEG) can range from about 6000 to about 10,000, from about 6000 to about 8000, from about 7000 to about 9000, from about 8000 to about 10,000. In a particular embodiment PEG with a molecular weight of about 8000 is used. In general, the presence of PEG provides a hydrophobic solution which forces hydrophilic nucleic acid molecules out of soltution. The advantages of using PEG which is a nondenaturing water soluble polymer, rather than an organic precipitating reagent (e.g., ethanol, isoproponal or phenol), are attributed to its benign chemical properties. According to the current invention, the PEG-induced nucleic acid precipitates are adsorbed to the surfaces of magnetically responsive microparticles which can be physically manipulated to facilitate the isolation of essentially pure species of nucleic acid molecules from complex solutions comprising mixtures of nucleic acids, in the presence or absence of other host cell biomolecules. Although numerous biological macrostructures (bacteriophage, ribosomes, plant and animal viruses, proteins and nucleic acids) are precipitable with PEG, the threshold concentration required varies for each macrostructure (Lis, Methods in Enzymology, 1980). This observation makes it possible to use the instant method to isolate nucleic acid molecules, not only from other nucleic acid molecules having a different molecular size, but also from other host cell biomolecules and biological macrostructures, each of which will have a distinct PEG threshold concentration at which it will precipitate.
Suitable salts which are useful for facilitating the adsorption of nucleic acid molecules targeted for isolation to the magnetically responsive microparticles include sodium chloride (NaCl), lithium chloride (LiCl), barium chloride (BaCl2), potassium (KCl), calcium chloride (CaCl2), magnesium chloride (MgCl2) and cesium chloride (CsCl). In a preferred embodiment, sodium chloride is used. In general, the presence of salt functions to minimize the negative charge repulsion of the nucleic acid molecules. The wide range of salts suitable for use in the method indicates that many other salts can also be used and suitable levels can be empirically determined by one of ordinary skill in the art.
As used herein, xe2x80x9cfacilitated adsorptionxe2x80x9d refers to a process whereby a precipitating reagent, (e.g., a poly-alkyelene glycol) is used to promote the precipitation and subsequent adsorption of a species of DNA molecules, which were initially in mixture, onto the surface of a solid phase carrier. The resulting reversible interaction is distinct from, for example, an interaction between two binding partners (e.g., streptavidin/biotin, antibody/antigen or a sequence-specific interaction), which are conventionally utilized for the purpose of isolating particular biomolecules based on their composition or sequence.
Suitable magnetically responsive paramagnetic microparticles have sufficient surface area to permit efficient binding and are further characterized by having surfaces which are capable of reversibly binding nucleic acids. Suitable solid phase carriers include, but are not limited to, other particles, fibers, beads and or supports which have an affinity for DNA and which can embody a variety of shapes, that are either regular or irregular in form, provided that the shape maximizes the surface area of the solid phase, and embodies a carrier which is amenable to microscale manipulations. Generally, paramagnetic microparticles are used.
As used herein, xe2x80x9cparamagnetic microparticlesxe2x80x9d refers to microparticles which respond to an external magnetic field (e.g., a plastic tube or a microtiter plate holder with an embedded rare earth (e.g., neodymium) magnet but which demagnetize when the field is removed. Thus, the paramagnetic microparticles are efficiently separated from a solution using a magnet, but can be easily resuspended without magnetically induced aggregation occurring. Preferred paramagnetic microparticles comprise a magnetite rich core encapsulated by a pure polymer shell. Suitable paramagnetic microparticles comprise about 20-35% magnetite/encapsulation ratio. For example, magnetic particles comprising a magnetite/encapsidation ration of about 23%, 25%, 28% 30% 32% or 34% are suitable for use in the present invention. Magnetic particles comprising less than about a 20% ratio are only weakly attracted to the magnets used to accomplish magnetic separations. Depending on the nature of the host cell, the viscosity of the cell growth and the nature of the vector (e.g. high or low copy) paramagnetic microparticles comprising a higher percentage of magnite should be considered. The use of encapsulated paramagnetic microparticles, having no exposed iron, or Fe3O4 on their surfaces, eliminates the possibility of iron interfering with polymerase function in certain downstream manipulations of the isolated DNA. However the larger the magnetite core the higher the chance of encapsulation leakage (e.g., release of iron oxides). Suitable paramagnetic microparticles for use in the instant invention can be obtained for example from Bangs Laboratories Inc., Fishers, Ind. (e.g., estapor(copyright) carboxylate-modified encapsulated magnetic microspheres).
Suitable paramagnetic microparticles should be of a size that their separation from solution, for example by magnetic means or by filtration, is not difficult. In addition, preferred paramagnetic microparticles should not be so large that their surface area is minimized or that they are not suitable for microscale manipulation. Suitable sizes range from about 0.1 xcexc mean diameter to about 100 xcexc mean diameter. A preferred size is about 1.0 xcexc mean diameter.
As used herein, the term xe2x80x9cfunctional group-coated surfacexe2x80x9d refers to a surface which is coated with moieties which reversibly bind nucleic acid (e.g., DNA, RNA or polyamide nucleic acids (PNA)). One example is a surface which is coated with moieties which each have a free functional group which is bound to the amino group of the amino silane or the microparticle; as a result, the surfaces of the microparticles are coated with the functional group containing moieties. The functional group acts as a bioaffinity adsorbent for polyalkylene glycol precipitated DNA. In one embodiment, the functional group is a carboxylic acid. A suitable moiety with a free carboxylic acid functional group is a succinic acid moiety in which one of the carboxylic acid groups is bonded to the amine of amino silanes through an amide bond and the second carboxylic acid is unbonded, resulting in a free carboxylic acid group attached or tethered to the surface of the paramagnetic microparticle. Suitable solid phase carriers having a functional group coated surface that reversibly binds nucleic acid molecules are for example, magnetically responsive solid phase carriers having a functional group-coated surface, such as, but not limited to, amino-coated, carboxyl-coated and encapsulated carboxyl group-coated paramagnetic microparticles.
One embodiment of the instant invention is a method of selectively isolating a target species of nucleic acid molecule, on the basis of its molecular size, from a solution comprising a mixture of the target nucleic acid species in the presence or absence of other species of nucleic acid molecules and other biomolecules. As described herein, the method comprises preparing a combination comprising a mixture of nucleic acids in the presence of polyethylene glycol (PEG) and salt, wherein the PEG and salt concentrations are sufficient to selectively precipitate a particular species of nucleic acid molecule, which has been targeted for isolation. The PEG and salt should be present at levels which are sufficient to precipitate the targeted nucleic acid species, but insufficient to precipitate relatively smaller sized nucleic acid molecules or other host cell biomolecules. The precipitated nucleic acid species targeted for isolation is removed from the solution by adding a solid phase carrier, such as a magnetically responsive microparticle, which has a functional group-coated surface that reversibly binds nucleic acid molecules to its surfaces. The nucleic acid-coated carrier represents a solid phase product which can subsequently be removed from the starting solution by the application of an external force (e.g., centrifugation, filtration or magnetic field).
The removal of the solid phase microparticle from the solution, results in the isolation of a target species of nucleic acid molecule, characterized by a particular molecular size, which is essentially free of other host cell biomolecules and as a consequence produces a solution from which nucleic acids characterized by a particular molecular size have been removed. An additional species of nucleic acid molecule (e.g., a second, third, fourth, etc.) characterized by having a relatively smaller molecular size can subsequently be isolated from the resulting solution by adding solid phase carriers, having a functional group-coated surface that reversibly binds nucleic acid molecules, to the solution (from which nucleic acid molecules of relatively higher molecular weight have been removed) in the presence of sufficient polyethylene glycol and salt to precipitate the relatively smaller species of nucleic acid molecule subsequently targeted for isolation. The resulting combination is maintained under conditions which favor the adsorption of the second nucleic acid species, but not other host cell biomolecules present in the solution, to the surfaces of the microparticles, thereby producing a second solid phase product. The removal of the second solid phase product from the solution results in the isolation of an additional species of nucleic acid molecule that is essentially free of other species of nucleic acid molecules, characterized by different molecular sizes, and of other biomolecules present in the starting solution.
As used herein a xe2x80x9chost cellxe2x80x9d is any cell into which exogenous DNA can be introduced, producing a host cell which contains exogenous DNA, in addition to host cell DNA. As used herein the terms xe2x80x9chost cell DNAxe2x80x9d and xe2x80x9cendogenous DNAxe2x80x9d refer to DNA species (e.g., genomic or chromosomal DNA) that are present in a host cell as the cell is obtained. As used herein, the term xe2x80x9cexogenousxe2x80x9d refers to DNA other than host cell DNA; exogenous DNA can be present into a host cell as a result of being introduced in the host cell or being introduced into an ancestor of the host cell. Thus, for example, a DNA species which is exogenous to a particular host cell is a DNA species which is non-endogenous (not present in the host cell as it was obtained or an ancestor of the host cell). Appropriate host cell include, but are not limited to, bacterial cells, yeast cells, plant cells and mammalian cells.
The term xe2x80x9clysed host cell suspensionxe2x80x9d, as used herein, refers to a suspension comprising host cells whose membranes have been disrupted by any means (e.g., chemical, such as alkali or alkali and anionic detergent treatment, isotonic shock, or physical disruption by homogenization), such a suspension is a mixture of host cell biomolecules, cellular components and disrupted membrane debris. In one embodiment, a lysed host cell suspension suitable for use in the instant invention is prepared by contacting host cells with an alkali and anionic detergent (e.g., sodium dodecyl sulphate (SDS)) solution (e.g., 0.2 N NaOH, 1% SDS). Optionally, lysozyme could be included in the lysis buffer. The presence of an anionic detergent in the lysing solution functions to produce an anti-protein environment by neutralizing the effective charge of the proteins, thereby minimizing their attraction to the surfaces of the functional group-coated paramagnetic microparticles. In one embodiment, the lysed host cell suspension is non-neutralized. Optionally, RNase can be added to the host cell lysate to degrade host cell RNA, thereby allowing DNA to bind to the magnetic microparticles free, or essentially free, from RNA. The necessity of including a Rnase step will largely be determined by the size of the nucleic acid species that is targeted for isolation in the particular PEG-induced precipitation that is being performed. For example, if the conditions selected for isolation are appropriate for isolating nucleic acids comprising at least 4000 base pairs, then it is unlikely that RNA species will be an appreciable contaminant.
In one embodiment, the present invention provides a method of selectively isolating a species of nucleic acid molecule present in a mixture from other nucleic acid molecules, and from other biomolecules, biological macrostructures, or reagents possibly present in the starting material. More specifically, this embodiment of the method involves: combining a mixture which comprises the target species of nucleic acid molecules to be isolated admixed with other nucleic acid molecules, biomolecules, biological macrostructures or reagents; solid-phase carriers (e.g., particles) having a functional group-coated surface, which acts as a bioaffinity absorbent for nucleic acids; a suitable concentration of a nucleic acid precipitating reagent(s) (e.g., polyethylene glycol (PEG) and salt, or an alcohol, such as ethanol or isopropanol) to result in the facilitated absorption of the target species of nucleic acid molecules, but not of smaller-sized species of nucleic acid molecules or other biomolecules, biological macrostructures or reagents present in the starting material. Separation of the target species of nucleic acid molecule is accomplished by applying an external force (e.g., magnetic field, centrifigation, filtration) suitable to remove the solid phase carrier having the selectively precipitated nucleic acid bound thereto from the combination. In a preferred embodiment the solid phase carrier is a paramagnetic microparticle and separation is accomplished by applying a magnetic field of appropriate strength. In a further embodiment the solid phase carrier is a paramagnetic microparticle and separation is accomplished by applying a magnetic field of at least 1000 Gauss. This embodiment of the invention is useful for example to isolate a restriction enzyme digest fragment having a particular molecular size from smaller fragments present in the same digest; for isolating a single PCR product from a multiplex PCR reaction; for the selection of DNA fragments having a homogenous sized distribution resulting from a shearing procedure (e.g., nebulizer, sonicator, hydroshear); for removing a nucleic acid template from a sequencing reaction or for selectively precipitating the extension products (e.g., Sanger Sequencing products) from a detemplated sequencing reaction prior to capillary electrophoresis. For example, the production of shattered DNA libraries for large scale sequencing experiments requires a size-selection step to minimize the deviation in size of the DNA inserts selected for cloning. The ability to produce a library comprising sheared DNA fragments, characterized by a narrow size distribution improves an investigator""s ability to construct a map of the original pre-sheared DNA molecule. Using the method described herein an investigator can preselect a cut off size and formulate a binding buffer appropriate to precipitate and selectively adsorb a homogeneous population of DNA fragments. This embodiment can also be used to isolate extension products from a detemplated sequencing reaction mixture. The adsorbed nucleic acid molecules (e.g., Sanger sequencing products) can be thoroughly washed free of salts (e.g., reagent) and excess terminals whose presence will interfere with the electrophoretic injection of the sample to be sequenced.
In a second embodiment, the method disclosed herein can be used to isolate two different species, for example, endogenous host cell nucleic acids and exogenous nucleic acid molecules, present in the starting material, by first isolating the relatively higher molecular weight host cell DNA, and subsequently isolating the relatively smaller-sized exogenous nucleic acid molecules. Thus, an alternative embodiment of the instant invention further provides a means for the selective removal of endogenous host cell DNA from a lysed host cell suspension by performing a first step designed to precipitate and promote the adsorption of host cell DNA chromosomal to the functional group-coated surfaces (e.g., a carboxyl-group-coated surface) of a suitable solid phase carrier (e.g., microparticle surfaces). The removal of the solid phase carrier (to which the host cell DNA is bound) from the resulting mixture results in the removal of the relatively larger-sized host cell DNA.
As described above, high quality exogenous DNA can subsequently be isolated from an exogenous DNA enriched supernatant by selectively precipitating and adsorbing the relatively lower molecular weight exogenous DNA to the surfaces of additional paramagnetic microparticles which are introduced resulting into the supernatant.
This alternative embodiment, which results in isolation of both endogenous host cell nucleic acid and exogenous nucleic acid (separately) comprises: combining functional group-coated paramagnetic microparticles and suitable concentrations of a precipitating reagent, for example, a polyalkylene glycol, and a salt to promote the facilitated adsorption of precipitated endogenous host cell nucleic acid (e.g., chromosomal DNA) and subsequently of exogenous nucleic acid molecules (e.g., bacterial or viral nucleic acids), each species being characterized by a particular molecular size, to the surfaces of the microparticles suspended therein; and the removal, such as by magnetic means, of the nucleic acid-coated microparticles from the resulting first combination. The removal of the microparticle having endogenous host cell nucleic acid adsorbed to its surfaces from the first combination results in the concomitant separation of host cell nucleic acid from both exogenous nucleic acid species and from other host cell biomolecules present in the sample. Exogenous nucleic acid present in the same sample can subsequently be isolated by producing a second combination by adding paramagnetic microparticles which have a functional group-coated surface and a sufficient quantity of a nucleic acid precipitating reagent to increase the concentration of the precipitating reagent to a level sufficient to result in the adsorption of exogenous nucleic acid to the microparticles suspended therein, thereby producing a third combination comprising exogenous nucleic acid bound to the microparticles; removing the paramagnetic microparticles from the third combination. Thus, exogenous nucleic acid bound to the microparticles is isolated from other host cell biomolecules present in the starting solution. Thus, the present invention also provides a method of selectively separating exogenous nucleic acids from relatively larger species of endogenous host cell nucleic acids present in the same sample.
The selective precipitation of endogenous host cell DNA (e.g., chromosomal or genomic DNA is mediated by concentrations of PEG as low as about 1% (w/v) and as high as about 4% (w/v) depending upon the size of the host cell DNA and the ionic strength of the solution. In a preferred embodiment, the concentration of PEG is preferably adjusted to about 3% (weight/volume). The subsequent selective precipitation of exogenous plasmid DNA is accomplished by adjusting the PEG concentration to a level which has been empirically determined to be optimal to promote the precipitation of a DNA species of a specified macromolecular size range. For example, exogenous DNA produced from the replication of a bacterial plasmid in a suitable strain of E. coli would be isolated by adjusting the PEG concentration of the second precipitation reaction to about 10% (weight/volume).
At high salt concentrations (e.g., synonymous with high ionic strengths) suitable paramagnetic microparticles will adsorb DNA fragments of all sizes. The term xe2x80x9chigh salt concentrationxe2x80x9d refers to salt concentrations greater than about 0.5M. At xe2x80x9clow salt concentrationsxe2x80x9d (or low ionic strengths), which as used herein connotes concentrations less than about 0.2 M, essentially no DNA, of any size, will be precipitated even in the presence of a PEG concentration that is as high as 12% (weight/volume) (Lis, John T, Methods in Enzymology 65: 437-353 (1980). At intermediate salt concentrations (e.g., ranging from about 0.3M to about 0.45M) the characteristic macromolecular size of the DNA species precipitated by a particular PEG concentration is a function of the interaction between ionic strength and PEG concentration and reflects a relationship between macromolecular size and requisite threshold PEG concentration required to precipitate DNA molecules of a given size. In general, smaller fragments of DNA will interact with the functional group-coated surfaces with a lower affinity than larger DNA fragments in the presence of relatively low concentrations of salt. To maximize yield and efficiency, sodium chloride concentration is preferably adjusted to about 0.55 M for the selective removal of host cell DNA from a lysed host cell suspension. Yields of bound DNA decrease if the salt concentration is adjusted to less than about 0.5 M or greater than about 5.0 M. Purity (e.g. quality) of recombinant DNA isolated during the second precipitation reaction decreases if the sodium chloride concentration exceeds about 0.55 M.
Another embodiment of the instant invention provides a method by which recombinant nucleic acid molecules expressed in host cells can be selectively isolated from host cell lysates comprising a mixture of nucleic acid molecules and other host cell biomolecules. The following is a description of this embodiment with reference to nucleic acid molecules as exemplified by DNA. It is to be understood that the instant embodiment is also useful for separation of other nucleic acids in a similar manner. This embodiment of the invention comprises the steps of: preparing a first combination comprising a lysed host cell solution prepared from cells expressing a recombinant nucleic acid; encapsulated carboxyl group-coated paramagnetic microparticles, and low percentage PEG and low molarity salt. According to the method disclosed herein, the PEG and salt are present at sufficient concentrations that high molecular weight host cell DNA is precipitated and reversibly binds (adsorbs) to the encapsulated carboxyl group-coated paramagnetic microparticles, thereby producing paramagnetic microparticles having host cell DNA bound thereto. The DNA-coated microparticles (and, thus, the microparticle-adsorbed endogenous DNA) are removed from the first combination, thereby producing a recombinant DNA-enriched supernatant. A second combination is produced by adding carboxyl group-coated paramagnetic microparticles to the recombinant DNA-enriched supernatant and sufficient polyethyleme glycol to result in the selective precipitation and adsorption of the relatively smaller sized recombinant DNA to the surfaces of the micro-particles, thereby producing paramagnetic microparticles having recombinant DNA bound thereto; and removing the paramagnetic microparticles (and thus, the adsorbed recombinant DNA), whereby recombinant DNA is selectively isolated from host cell DNA.
Examples of recombinant DNA which can be introduced into a host cell include, but are not limited to, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), PACs, P1s, cosmids and bacterial plasmids. The exogenous DNA may be directly introduced into a host cell, or an ancestor thereof, by means well known to one of ordinary skill in the art, such as transformation or transfection methods. Alternatively, plasmid DNA may be indirectly introduced into a host cell, or its ancestor by use of a phage into which exogenous DNA has been packaged. Suitable plasmid DNAs which can be packaged into a phage include a cosmid or P1 vector. Suitable host cells include bacterial cells, yeast cells, plant cells and mammalian cells. For example, suitable strains of E. coli bacteria include but are not limited to: DH5xcex1, DH1, DH10B, DH12S, C600 or XL-1 Blue. As used herein the term xe2x80x9cplasmidxe2x80x9d refers to double stranded circular DNA species which originate from an exogenous source (e.g., are introduced into a host cell) and which are capable of self-replication independent of host chromosomal DNA. Thus, the term encompasses cloned DNA produced from the replication of any of the above-mentioned vectors. Suitable vectors are well known in the art and include, for example high copy vectors, selected from, but not limited to the group consisting of pUC, pOT, pBluescript, pGEM, pTZ, pBR322, pSC11, pACYC, SuperCos and pWE15.
BACs are particularly difficult to separate and purify from cleared lysates due to their low concentrations in the lysates, which is attributable to their low copy number presence in the host cell. However, BAC DNA (e.g., up to 180 kb in size) is readily isolated by the method of the present invention. Cosmids are particularly difficult to isolate from expression host cells using commercially available chromatography-based methods because of their relatively large size (e.g., 35 to 40 kb). However, cosmids are readily separated by the methods of the present invention.
Thus, the method of the present invention is also useful to separate recombinant DNA resulting from the replication of an exogenous vector from a host cell lysate containing an admixture of host cell biomolecules, including host cell DNA and exogenous cloned DNA produced by the host cell. Yields of recombinant DNA following elution typically approach 100% when the magnetic microparticles are used in excess. High copy plasmid DNA templates have been prepared according to the disclosed method characteristically result in sequences which were read with 99% of unedited accuracy. The simplicity and robust nature of the disclosed method makes it particularly useful for the preparation of DNA sequencing templates for automated nucleotide sequencing.
Another embodiment of the present invention relates to a method of isolating a nucleic acid molecule suitable for use as a template for nucleotide sequencing using either manual or high-throughput automated sequencing methods. This embodiment comprises: treating host cells with an alkali and anionic detergent combination, thereby producing a lysed host cell suspension; suspending paramagnetic microparticles which bind DNA in the lysed host cell suspension in the presence of low percentage polyethylene glycol and low molarity salt, which promotes the precipitation and selective adsorption of host cell DNA to the surfaces of the microparticles; removing the paramagnetic microparticles having host cell DNA bound thereto from the suspension, thereby producing a plasmid-enriched supernatant; combining additional paramagnetic microparticles which bind DNA with the plasmid-enriched supernatant and adjusting the polyethylene glycol percentage and salt concentration of the supernatant to levels which result in binding of plasmid DNA to the microparticles; removing the microparticles having plasmid DNA bound thereto from the supernatant; washing the microparticles with a wash buffer to remove impurities adsorbed to the microparticles, thereby producing a purified template; and contacting the microparticle-bound purified template with an elution buffer, whereby the plasmid DNA template is released from the microparticles and is dissolved in the elution buffer, thereby isolating a purified plasmid DNA template suitable for nucleotide sequencing.
The present invention also further relates to a kit comprising reagents for preparing a host cell lysate which is appropriate for automated processing according to a system which has been optimized for high through-put DNA templates preparation; and an aqueous solution of paramagnetic microparticles having a functional group-coated surface capable of reversibly binding nucleic acid molecules. In addition the kit may comprise either at least one binding preformulated binding buffer comprising polyalkylene glycol and salt, at concentrations which been empirically determined to be appropriate to reversibly bind nucleic acid molecules characterized by a particular molecular size range onto the functional group-coated surfaces of the paramagnetic microparticles, or reagents for the formulation of a binding buffer. Additionally, the kit may comprise at least one preformulated high ionic strength buffer suitable for use as a wash buffer, or reagents for preparing such buffer; a preformulated elution buffer or reagents for its preparation; and a magnetic microtiter plate holder designed to optimize features of the magnetic field known to be crucial to the efficiency of automated processing. The design of the magnetic plate holder is instrumental in producing a magnetic field having the requisite uniformity and field strength to maximize the efficiency achievable with automatic processing. Field Strength of up to and over 1600 Gauss can be achieved with the use of N35 rare earth magnets configured with alternating North and South polar spaced 9 mm apart. Since the magnetically responsive microparticles are paramagnetic they will attract to either pole.
The isolation of high quality nucleic acid preparations from starting solutions of diverse composition and complexity is a fundamental technique in molecular biology. Thus, as a result of the work described herein, novel and readily automatable methods of isolating and purifying nucleic acid molecules are now available. Nucleic acids isolated by the disclosed method can be used for molecular biology applications requiring high quality nucleic acids, for example, the preparation of DNA sequencing templates, the microinjection, transfection or transformation of mammalian cells, the in vitro synthesis of RNA probes, reverse transcription cloning, cDNA library construction, PCR amplification, or gene therapy research, as well as for other applications with less stringent quality requirements including, but not limited to, transformation, restriction endonuclease or microarray analysis, selective RNA precipitations, in vitro transposition, separation of multiplex PCR amplification products, preparation of DNA probes and primers and detemplating protocols.
The following Examples are offered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachings of all references cited herein are hereby incorporated, in their entirety, by reference.
Beads
The paramagnetic microparticles used in the following examples were polymer p (S/V-COOH)Mag/Encapsulated paramagnetic microspheres (brown, mean diameter 1.12 xcexcm, 10% solids; catalog number ME03N) from Bangs Laboratories, Inc., (Fishers, Ind.). The particles were stored in phosphate buffered saline (PBS) at a concentration of 20 mg/ml.
Bead Pretreatment
Prior to use in any of the separation protocols exemplified herein, all beads are washed four times with 10 mM Tris Acetate pH 7.8, and diluted tenfold in the 10 mM Tris Acetate buffer. More specifically, all beads are washed and diluted according to the following protocol:
1. Place 4 mls of beads into a tube.
2. Add 26 mls of 10 mM Tris Acetate.
3. Apply a magnet to the suspension, and pour off the wash buffer.
4. Repeat steps 2-3, 3 times.
5. Resuspend the washed beads in a final volume of 40 mls of 10 mM Tris Acetate.
Lysis Buffer Preparation
Prepare 0.8 N NaOH/8% SDS lysis buffer by combining 160 ml of 2 N NaOH, 160 mls of 20% SDS, and 80 mls of distilled water. Immediately after mixing the buffer should be warmed to, and maintained at a temperature of 60-70 degrees C. Lysis buffer should be prepared fresh daily.
Sample Preparation
Pellet cells (e.g., centrifuge at 1645xc3x97g, for 10 mins.); decant culture medium; resuspend the pelleted cells in water in a final volume that is appropriate based on the density of the starting culture; vortex (e.g., 1200 rpm for 6 mins); and transfer an aliquot of the resulting cell suspension to flat bottom multiwell plates (e.g., 150 xcexcl in a 96-well plate).
Ethanol Wash Protocol
The paramagnetic microparticle bound nucleic acid species is washed free of contaminants using three to five sequential washes with 70% Ethanol, with the volume transfers either being accomplished by hand or by using an automated plate washer (e.g., Tecan SLT/PW). A suitable wash protocol using an automated plate washer would consist of the three to five repetitions of the following cycle: add 250 ul ethanol, soak for 20 seconds, aspirate for 7 seconds and repeat.
Sequencing
Suitable automated sequencing reactions can be performed using Amersham ET primer Thermosequence 2 terminator chemistry, Big Dye primer and terminator chemistries, NEN/Omnibase terminator chemistries, or AB1 FS terminator chemistry using established protocols that are well known to those of skill in the art. Sequence results can be obtained using AB1377 Sequencers, MD MegaBACE capillary or ABI 3700 capillary sequencers. All agarose gels were run using 1% final agarose (U.S. Biochemical #32827) with 1xc3x97TBE buffers. The field strength was 10 V/cm with run times from 40-60 minutes. The gels were post-stained with ethidium bromide and visualized under UV.